PROJECT SUMMARY/ABSTRACT Many leading human health organizations such as the World Health Organization and the Centers for Disease Control and Prevention (CDC) have declared that the increased prevalence of bacterial pathogens that are resistant to multiple antibiotics is a significant human health crisis. The emergence of these multi-drug resistant (MDR) pathogens is largely due to the sharing of resistance genes by plasmid mediated horizontal gene transfer. Bacterial plasmids are mobile genetic elements that can confer resistance to a variety of antibiotics, including those that are considered to be ?drugs of last resort?. Our long-term goal is to aid the development of strategies that can slow the spread of antibiotic resistance by gaining insight into the co-evolutionary processes that allow bacteria to improve the persistence of newly acquired MDR plasmids. Newly acquired resistance plasmids often do not persist in the absence of antibiotics, but we and others have shown that single mutations in the bacterial host, the plasmid, or both can rapidly improve this persistence. We and others also identified critical mutations in chromosomally encoded accessory helicases. Plasmid-helicase interactions in bacteria may therefore be key to the ability of bacterial pathogens to retain newly acquired MDR plasmids. Unfortunately, the molecular mechanisms that explain the positive effects of these mutations on plasmid persistence are unknown. Importantly, we also showed for the first time that these mutations pre-adapt the bacteria to other MDR plasmids that they acquire later in time, leading to their enhanced persistence (referred to as increased plasmid permissiveness). This suggests that bacteria with increased permissiveness can serve as stable repositories for multiple MDR plasmids, eventually generating strains with an expanded arsenal of resistance genes. This possibility has never been tested. Using molecular techniques, experimental evolution and mathematical modeling, we propose to test the following hypotheses: (i) chromosomal mutations can pre- adapt bacteria to other plasmids, leading to greater plasmid permissiveness; (ii) plasmid permissiveness can expand the spectrum of antibiotic resistance traits within a bacterial species; and (iii) accessory helicases are linked to the persistence of newly acquired MDR plasmids across a wide spectrum of bacterial pathogens. This will be done through achieving the following Specific Aims: (1) Test the generality of (i) increased plasmid permissiveness after host/plasmid coevolution, and (ii) helicase mutations as a mechanism of host adaptation to novel MDR plasmids.; (2) determine the effects of plasmid persistence and permissiveness on the emergence of expanded drug resistance; (3) determine the molecular mechanism of plasmid cost amelioration resulting from mutations in accessory helicases. If our hypotheses are supported by our data, mutations that stabilize one plasmid could lead to improved persistence of other plasmids, and expand the arsenal of resistance genes in the same cell. Our findings will aid the development of new therapies aimed at slowing down the spread of antibiotic resistance in bacterial pathogens.!